CELL-SPECIFIC EXPRESSION OF modRNA

ABSTRACT

Disclosed is an expression regulatory system for cell-specific transcription (expression) of a protein of interest, for example a cell cycle inducer that reactivates proliferation in adult or neonatal cardiomyocytes or insulin-producing beta cells. The expression regulatory system comprises a first nucleic acid that encodes a microRNA recognition element that specifically binds a target cell miR, and a translation suppressor protein; and a second nucleic acid that comprises a suppressor protein interaction motif that binds the translation suppressor protein, and a gene that encodes a protein of interest. When a cell of interest is co-transfected with the first and second nucleic acids of the system, the protein of interest expressed in a cell-specific fashion.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/354,814, filed Mar. 15, 2019, which issued as U.S. Pat. No.11,299,749 on Apr. 12, 2022, which was a continuation ofPCT/US2017/052035 filed Sep. 18, 2017 and published on Mar. 22, 2018 asWO 2018/053414, which claims priority to U.S. provisional applicationNo. 62/395,701 filed Sep. 16, 2016, the contents of which are herebyincorporated by reference into the present application.

SEQUENCE LISTING

The instant application contains a Sequence Listing, created on Feb. 28,2022; the file, in ASCII format, is designated H2277405.txt and is 17.9kilobytes in size. The file is hereby incorporated by reference in itsentirety into the instant application.

TECHNICAL FIELD

The present disclosure relates generally to a platform for thecell-specific expression of therapeutic proteins in vitro, ex vivo andin vivo, using a cell-specific transcriptional regulatory system basedon cell-specific miR override of gene expression suppression.

BACKGROUND OF THE DISCLOSURE

Chemically modified messenger RNA (modRNA) is a therapeutic strategythat enables the cellular machinery to produce genes of interest withoutmodifying the genome. Thus, modRNA avoids several of the problems thathave arisen with conventional gene therapy, including lack of genomicintegration, persistence of expression, immunogenicity, difficulty inscalability and production, need for life-long monitoring fortumorigenesis and other adverse clinical outcomes, and the potential forvector escape into the systemic circulation and long-term expressionelsewhere in the body.

modRNA has considerable potential as a therapy for disease. Delivery ofa cell cycle inducer via modRNA, for example, would trigger growth ofbeta cells in individuals with diabetes or restore proliferation ofcardiomyocytes following myocardial infarction or heart failure.Diabetic neuropathy may be lessened by the ability to deliver genesencoding nerve growth factor. Additionally, with the advent of genomeediting technology, CRISPR/Cas9 or transcription activator-like effectornuclease (TALEN) transfection will be safer if delivered in a transientand cell-specific manner.

However, none of the available transfection reagents for modRNA offersboth a high level of gene expression and the ability to target any cellof interest. For example, a common in vivo transfection reagent is invivo-jetPEI® (Polyplus-transfection® SA, Illkirch, France), which is apolymer based reagent that complexes with modRNA to form nanoparticles.However, in vivo-jetPEI primarily targets lung tissue in vivo andsignificantly lowers transfection efficacy compared to naked modRNA.

Therefore, what is needed is a modRNA-based gene delivery system thatachieves a high level of gene expression exclusivity in a cell ofinterest.

SUMMARY OF THE DISCLOSURE

The present disclosure provides an expression regulatory platform forcell-specific transcription based on the exploitation of a repressorRNA-binding protein/k-motif interaction coupled with cell-specific miRoverride of the repressor function to control expression of a deliveredmodRNA in a cell-specific fashion. RNA-binding proteins such as thearchaeal protein L7Ae and eukaryotic homologs thereof such as L30erecognize a distinctive RNA motif, the kink-turn (k-turn or k-motif asreferred to herein). By incorporating the k-motif into a first constructthat encodes a gene of interest (GOI) and including a recognitionelement for a cell-specific miR in a second construct that encodes theRNA-binding protein, suppression of expression of the GOI is overriddenwhen the two constructs are co-transfected into the appropriate celltype. The platform incorporates modified mRNA

The present disclosure, therefore, relates to a method for achievingcell-specific expression of a modRNA of a gene of interest (GOI) theexpression of which is desired only in the cell of interest. In oneaspect, the disclosure describes an expression regulatory system forcell-specific transcription, the system comprising a first nucleic acidthat encodes (1) a cell-specific microRNA (miR) recognition element, and(2) a translation suppressor protein; and a second nucleic acid thatencodes (1) a suppressor protein interaction motif, for example aK-motif, downstream of its 5′UTR that binds the translation suppressorprotein, and (2) a gene that encodes a protein of interest. The nucleicacids are modRNA.

By swapping out the miR recognition element, cell specificity can bemodulated, making the system adaptable to other cell types.

In another aspect, the present disclosure relates to short-termexpression of cardiomyocyte (CM)-specific modRNA of candidate genes,such as cell cycle inducer genes, the expression of which reactivates CMregeneration, which is important following post-myocardial infarction orin heart failure settings. The method is based on the observation thatcell cycle inducer genes, for example, Lin28 and Pkm2, delivered asmodRNA using the cell-specific delivery system of the disclosurefollowing MI significantly induces CM and non-CM proliferation. Sinceincreased non-CM proliferation can lead to enhanced cardiac scarring, itwas necessary to develop a CM-specific modRNA that allows expression ofgenes only in cardiomyocytes.

The present disclosure describes CM-specific modRNA that allows modRNAtranslation exclusively in CMs. In one embodiment, CM-specific Lin28 orPkm2 modRNA expression results in significant CM proliferation withoutsignificantly changing non-CM proliferation. In another embodiment,based on CM-specific modRNA, a novel lineage tracing adult mouse modelthat is based on co-expression destabilized Cre recombinase andcandidate genes in Rosa26^(tdTomato) using CM-specific modRNA wasdeveloped.

In one aspect, the disclosure relates to an expression regulatory systemfor cardiomyocyte-specific expression comprising a first nucleic acidthat encodes a recognition element for microRNA (miR recognitionelements serve as an anti-miR approach) that binds specifically to atarget cardiomyocyte miR, and prevents the translation of a suppressorprotein (L7Ae); and a second nucleic acid that comprises a gene ofinterest and a kink-turns motif (K-motif) that are bound by thesuppressor protein (L7Ae). Binding of L7Ae to the K motif inhibits theexpression of the genes that had the K motif.

In one embodiment of the translational regulatory system, the targetcardiomyocyte miR is selected from the group consisting of miR1, miR29,miR126, miR133a, miR199, miR208a and miR378. In another embodiment, thetarget cardiomyocyte miR is selected from the group consisting of miR1,miR 208a and miR1 in combination with miR208a.

In one embodiment of the expression regulatory system, the suppressorprotein is L7Ae and the protein interaction motif is K-motif. L7Ae is anRNA binding protein that represses translation of the targetedtranscript. L7Ae targets a specific sequence called the k-motif ork-turn. Accordingly, the k-motif is built into the nucleic acid of thepair that encodes the GOI. Ordinarily, when the other nucleic acid ofthe pair that encodes L7Ae is expressed normally, L7Ae is able to bindto the k-motif, thereby repressing expression of the GOI encoded by thatnucleic acid.

In an embodiment of the present system, the nucleic acid encoding L7Aealso contains a cell-specific miR recognition element. When expressed inthe appropriate cell, cell-specific miR binds the miR recognitionelement to halt expression of L7Ae, eliminating suppression of the GOIon the other nucleic acid.

In one embodiment of the translational regulatory system, the protein ofinterest is a reporter protein or other gene of interest. In oneembodiment of the translational regulatory system, the reporter proteinor selection marker is a fluorescent protein, an antibiotic resistancemarker or other gene of interest. In one embodiment of the translationalregulatory system, the reporter protein or selection marker is selectedfrom the group consisting of green fluorescence protein (GFP), inactivehuman CD25 (ihCD25). In one embodiment of thetranscriptional/translational regulatory system of the disclosure, theprotein of interest is a cell cycle inducer protein. In one embodimentof the translational regulatory system, the cell cycle inducer proteinis selected from the group consisting of Lin28, Pkm2, and Cyclin D2. Inone embodiment of the transcriptional regulatory system, said firstnucleic acid comprises the nucleotide sequence of SEQ ID NO: 2, SEQ IDNO: 3, or SEQ ID NO: 4. In one embodiment of the transcriptionalregulatory system, said second nucleic acid comprises the nucleotidesequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.

In one aspect, the disclosure relates to a composition comprising firstand second modified RNAs (modRNAs), wherein said first modRNA is anexpression product of the first nucleic acid of claim 1, 2 or 3 and thesecond modRNA is an expression product of the second nucleic acid.

In one aspect, the disclosure relates to a method for expressing aprotein in cardiomyocytes (CMs), the method comprising contacting saidCMs with a modRNA encoding an miR recognition element specific for acardiomyocyte miR target, wherein the modRNA comprises the nucleotidesequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 3.

In one aspect, the disclosure relates to a vector comprising first andsecond nucleic acids as described herein.

In one aspect, the disclosure relates to a transcriptional/translationalregulatory kit comprising the first and second nucleic acids asdescribed herein or a vector comprising first and second nucleic acidsas described herein.

In one aspect, the disclosure relates to a method forinducing/reactivating proliferation of cardiomyocytes followingmyocardial infarction (MI), the method comprising contacting saidcardiomyocytes or a portion of said myocytes with a first modRNA thatencodes a cardiomyocyte-specific miR and a second modRNA that encodes acell cycle inducer gene.

In one aspect, the disclosure relates to the disclosed method, whereinthe cell cycle inducer gene is selected from the group consisting ofLin28, Pkm2 and Cyclin D2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plasmid map of pTEMPLZ used in generating the modRNA ofthe disclosure.

FIG. 2 shows in graphic (top panel) and table form (bottom panel) thePCR settings for synthesizing DNA tailed template. Shown in box is theelongation step that must be set based on the size of the sequenceinsert. Elongation step requires 30 sec per KB of ORF insert. PCRsetting is based of manufacturer instructions from 2×KAPA HiFi HotStartReadyMix kit.

FIGS. 3A-3C show the results of the quality control analysis for modRNAsynthesis. A 1% agarose gel determining correct size of the plasmidpTEMPLZ with ORF insert and tailed DNA template for IVT. B IdealNanodrop result of final modRNA product. Ideal concentration is between15-20 ug/ul. 260/280 values closer to 2 indicate purity. C Bioanalyzerresult for quality control of synthesized modRNA.

FIGS. 4A-4C A Whole heart view of mouse heart injected in vivo withmodRNA encoded with LacZ gene. 24 hours after injection, mouse wassacrificed, the heart was fixed with 4% PFA, and stained with x-gal. Bimmunostaining of mouse heart injected in vivo with modRNA encoded withnuclear GFP. (left) Cardiomyocytes (TropT: Red), Endothelial cells(Pecam1: Red) and smooth muscle cells (smMHC: Red) positive for nuclearGFP (Green). (DAPI: Blue). C cross section of Rosa26 LacZ mouse heartinjected with modRNA encoded with Cre Recombinase. Transfected cellswith Cre Recombinase can be stained with x-gal resulting in dark bluecolor.

FIGS. 5A and 5B shows adult mouse myocardial infarction and heartfailure models. Adult mouse myocardial infarction model (MI model) isperformed using a permanent ligation of left anterior descendingcoronary artery (LAD) following direct intramuscular injection ofmodRNA. One or more days post MI, hearts are collected and used forimmunostaining. B Adult mouse heart after MI is highly transfected withLuc; LacZ and nGFP modRNAs. A Several cell types are transfected withmodRNA, including cardiomyocytes (CM), cardiac fibroblasts (CF) andendothelial cells (EC).

FIGS. 6A-6H show that Pkm2 expression in adult CMs induces proliferationafter MI. A Relative expression of Pkm2 measured by qRT-PCR in mice'hearts 1 or 10 days after birth. B Experimental plan for immunostainingof Pkm2 or α-Actinin (CMs marker) at different stages of mouse heartdevelopment. C Representative images of Pkm2 expression at differentstages of mouse heart developmental. D pharmacokinetics of Pkm2expression post modRNA delivery in vivo. E Experimental timeline formeasuring the effect of Pkm2 on CMs proliferation F A Representativeimage of DNA synthesis (BrdU⁺) in CMs (α-Actinin⁺) and non-CMs cells(α-Actinin⁻) 7 days post-MI. G-H Quantification of hallmarkproliferation markers in CMs F or non-CMs G in adult mice 7 dayspost-MI. Results represent 2 independent experiments (n=4); white arrowheads point to CMs; yellow arrow heads point to non-CMs, ***, P<0.001,**, P<0.01, two-tailed student t-test, Scale bar 10 μm.

FIGS. 7A-7K show the design and function of _(cms)modRNA in vivo. AConstruct design and experimental timeline used to identify _(cms)miRs.B Immunostaining images of ihCD25 modRNA expression (red) with orwithout recognition elements for different miRs post transfection. CQuantification of the experiment in c. D modRNAs constructs design usedfor _(cms)Cre or _(cms)nGFP modRNAs delivery in vivo. E-F nGFP-K modRNA(green) transfected alone or co-transfected miR1-208, 4 days post-MI. ERepresentative images of hearts 7 days post-MI. F Transfectionefficiency with different ratios of nGFP-K and miR1-208. Rosa26^(mTmG)mice co-transfected with Cre-K+miR1-208. G Co-transfection ofCre-K+miR1-208. Red: Troponin I. H Quantification of the experiment ing. I Experimental timeline for evaluation of _(cms)Pkm2 modRNA effect onproliferation. Quantification of hallmark proliferation markers in CMs Jor non-CMs K 7 days post-MI. Results represent 2 independent experiments(n=3 mice); ****, P<0.0001, ***, P<0.001, **, P<0.01, N.S, NotSignificant, two-tailed student t-test (f) or One-way ANOVA, Bonferronipost-hoc test (j,c,k). Scale bar 10 μm or 50 μm in c and for h,respectively.

FIGS. 8A-8N show that _(cms)Pkm2 modRNA improves cardiac function andoutcome post MI. A Experimental timeline to evaluate cardiac functionand outcome. B MRI assessments of left ventricular systolic function 1month post-MI. Images depict left ventricular chamber (outlined in red)in diastole and systole. C Percentage of ejection fraction for theexperiments in b. D Echo evaluation of delta in percentage offractioning shorting differences between day 2 (baseline) and day 28post-MI. E Representative pictures of masson trichrome staining toevaluate scar size 28 days post-MI. F-I Quantification of scar size F,heart weight to body weight ratio G, CMs size H, and capillary density Imeasured 28 days post-MI. J-M Number of CMs with different treatments 28days post-MI. J Representative image of the number of CMs in each group.K Quantification of the experiment in j. L Representative images ofnuclei of isolated CMs (mono, bi or multi). M Quantification of theexperiment in I N Long-term post-MI survival curve for mice injectedwith Pkm2-K or luc-K modRNAs and co-transfected with miR1-208. Resultsrepresent 2 independent experiments (n=5 mice); ***, P<0.001, **,P<0.01, *, P<0.05, One-way ANOVA, Bonferroni post hoc test. P-values forlong term survival were calculated using the Mantel-Cox log-rank test.Scale bar 10 μm.

FIGS. 9A-9M show a lineage tracing of CMs expressing cms Pkm2 post-MIand shows increased number of transfected CMs and induction of keydownstream mediators of Pkm2's functions. A Experimental timeline usedfor cardiac lineage tracing in R26mTmG mice B Transfection efficiency (%GFP⁺) of CMs or non-CMs 28 days post-MI. C Representative images of CMsand their progeny (GFP⁺) 28 days post-MI. D Quantification of GFP⁺ CMs 3or 28 days post-MI. Ratio of heart to body weight E, relative size ofGFP⁺ CMs F, and number of nuclei in GFP⁺ CMs G in hearts, 28 dayspost-MI. H Representative image of GFP⁺ CMs, pH3⁺ or Ki67⁺ 28 dayspost-MI. Quantification of GFP⁺ pH3⁺ CMs I or GFP⁺ Ki67⁺ CMs J 28 dayspost transfection with _(cms)Luc or _(cms)Pkm2 with _(cms)Cre modRNA inMI model. K-M 2 days post-MI and administration of _(cms)ihCD25 with_(cms)Luc or _(cms)Pkm2 modRNAs, adult CMs were isolated using magneticbeads. K qRT-PCR analysis to validate purity of isolated CMs. L Geneexpression comparisons of key genes for both PPP (G6PD) and keydownstream indirect transcriptional targets of Pkm2 in adult CMs. MExpression of cell-cycle promoting genes or cell-cycle inhibitors.Results represent 2 independent experiments (n=3 mice); ***, P<0.001,**, P<0.01, *, P<0.05, N.S, Not Significant, two-tailed student t-test(b-j) or ANOVA with Bonferroni post hoc test (k-m). Scale bar 50 or 10μm in c or h, respectively.

FIG. 10A-10B shows that adult CMs are successfully transfected withmodRNA in vitro. Isolated adult CMs were transfected with nuclear GFP(nGFP) and imaged 20 hours post transfection (bar=10 μm.)

FIG. 11 is a bar graph showing that activation of proliferation of adultCMs in vivo using cell cycle inducer modRNAs do not compromise CMintegrity. CM size was measured using wheat germ agglutinin (WGA)staining for CMs cross-section area evaluation in hearts 7 days after MIwith different modRNA treatments. Results indicate no significantdifferences in CM integrity and size when cell cycle inducer Lin28 orPkm2 modRNAs were delivered in adult mouse MI model. Results representtwo independent experiments with n=3 mice, N.S, not significant,two-tailed student t-test.

FIGS. 12A-12C show that activation of proliferation of adult CMs in vivousing cell cycle inducer modRNAs reduces CM apoptosis and increasescapillary density. CM proliferation (Ki67+) apoptosis (TUNEL+) andcapillary density (Cd31+ luminal structures) were measured in the leftventricle of hearts 7 days post MI and different modRNA treatments. Arepresentative data showing Lin28, cell cycle inducer modRNA treatment 7days post MI induce CM proliferation, reduce apoptosis and increasecapillary density. Quantifiable results for apoptosis B or capillarydensity C level with the different treatments. Results indicatesignificant reduction in CM apoptosis and elevation in capillary densityusing cell cycle inducer genes such as Lin28 (red), and PKM2. Resultsrepresent two independent experiments with n=3 mice, ***, P<0.001,two-tailed student t-test.

FIGS. 13A-13C show that miR-1 and miR-208 are expressed exclusively inrat neonatal CMs in vitro. A Inactivate human CD25 (ihCD25) modRNA withor without miR recognition element for miR-208, miR1, miR133a, miR126,miR199, miR378 and miR29a were transfected into neonatal CM in vitro. 20hours post transfection cells were fixed and stained with anti CD25(red) and Troponoin I (CM marker, green). B Images taken for differenttreatments showing that when ihCD25 modRNA had recognition elements ofmiR-1 or miR-208, CMs (Troponoin I+ cells) were unable to translateihCD25 modRNA (Troponoin I+ and ihCD25+ CMs), other treatments resultedin ihCD25 translated in CMs. This indicate that only miR-1 or miR-208are CMs specific. C quantification of the experiments. Results representtwo independent experiments with n=3 wells, bar=10 mm.

FIGS. 14A-14C show that miR-1 and miR-208 are expressed exclusively inrat neonatal CMs in vitro. A ihCD25 modRNA with or without miRrecognition element for miR-208 or miR1 were co-transfected with nGFPinto neonatal CM in vitro. 20 hours post transfection cells were fixedand stained with anti CD25 (red) and Troponin I (CMs marker, greennuclear). nGFP was used as transfection control. B images taken fordifferent treatments showing that when ihCD25 modRNA had recognitionelements of miR-1 or miR-208, CMs (Troponoin I+ cells) were unable totranslate ihCD25 modRNA (Troponoin I+ and ihCD25+ cells), however ihCD25modRNA without miR recognition elements was able to translate in CMs.all cells were transfected with nGFP indicating that modRNA wasdelivered successfully. C quantification of the experiments. Resultsrepresent two independent experiments with n=3 wells, bar=10 mm.

FIGS. 15A-15C show that miR-1 and miR-208 express exclusively in adultmouse heart in vivo. A ihCD25 modRNA with or without miR recognitionelement for miR-208 or miR1 were co-transfected with nGFP into adultmouse heart in MI model. 20 hours post MI and delivery of modRNA heartswere collected, fixed and stained with anti CD25 (red), Troponoin I (CMsmarker, white) and nGFP (green). nGFP was used as transfection control.B images taken for different treatments showing that when ihCD25 modRNAhad recognition elements of miR-1 or miR-208, CMs (Troponoin I+ cells)were unable to translate ihCD25 modRNA (Troponoin I+ and ihCD25+ cells),transfecting ihCD25 without miR recognition site resulted in ihCD25translated in CMs. all cells were transfected with nGFP indicating thatmodRNA was delivered successfully. C quantification of the experiments.Results represent two independent experiments with total n=5 mice,bar=10 mm.

FIGS. 16A-16C show that nGFP CMs specific modRNA carrying recognitionelement for miR-208, miR-1, or both, in 1:1 ratio, show nGFP translationmostly in CMs in vitro. A CMs specific modRNA design. B nGFP CMsspecific modRNA caring recognition element for miR-208, miR-1, or both,in 1:1 ratio, were transfected into neonatal rat CMs in vitro. 20 hourspost transfection cells were fixed and stain for nGFP (nuclear green)and Troponin I (CM marker, red). C quantification of the experimentdescribed in B. Results represent two independent experiments with n=3wells, bar=10 mm.

FIGS. 17A-17C show nGFP CMs-specific modRNA carrying recognition elementfor miR-208, miR-1, or both, in 1:2.5 ratio or higher, show nGFPtranslation exclusively in CMs in vitro. A CMs specific modRNA design. BnGFP CMs specific modRNA caring recognition element for miR-208, miR-1,or both, in different ratios, were transfected into neonatal rat CMs invitro. 20 hours post transfection cells were fixed and stain for nGFP(green nuclear) and Troponin I (CM marker, red). C quantification of theexperiment described in B. Results represent two independent experimentswith n=3 wells, bar=10 mm.

FIG. 18 nGFP CM-specific modRNA carrying recognition element for bothmiR-1 and miR-208, in 1:0.5 ratio or higher, show nGFP translationexclusively in CMs in vivo. Results represent two independentexperiments with n=3 mice, bar=10 mm.

FIGS. 19A-19C Pkm2 CM-specific modRNA promotes proliferation exclusivelyin CMs. A Experimental time line. B modRNA design used in theexperiments. C Lin28/PKM2 CMs-specific modRNA promotes proliferationexclusively in CMs. PKM2 modRNA carrying a k motif co-transfected withL7AE-miR1 and miR208 were tested for reactivation of adult mousecardiomyocytes proliferation 7 days post-delivery in a mouse myocardialinfarction model. Red dashed line represents control proliferation rate.Results represent 2 independent experiments with n=2 mice (total n=4mice), ***P<0.001, N.S., Not significant; two-tailed student t-test.

FIG. 20 L7AE modRNA did not elevate immune response in adult mousemyocardial infraction model. Quality images showing that no elevation inimmune response (CD45+ cells, red) can be seen after L7AE is beendelivered with or without recognition elements of different miRs. Scalebar=10 μm.

FIGS. 21A-21C Evaluation of L7AE modRNA with or without miR recognitionelement for miR-208 or miR1 or both on CMs proliferation and size, invivo. A Mouse adult heart after MI was injected with Luc, L7AE withoutmiR recognition element or with recognition element for miR-1 or miR-208or both. 7 days post MI hearts were collected, fixed and stain fordifferent proliferation markers such as Ki67, BrdU, H3P and Aurora B Band WGA for measuring CMs size in the treated hearts C Results indicatethat L7AE with miR recognition element for miR-208 or miR-1 or bothinduce CM proliferation without compromising CMs size. Results representtwo independent experiments with n=3 mice, N.S, not significant, ***,P<0.001, two-tailed student t-test.

FIGS. 22A-22C show the transcriptional/translational regulatory systemused to express nGFP.

FIGS. 23A-23D show the results of isolation of transfected adult CMsfrom heart post MI using a CMs specific modRNA approach and magneticbead sorting. A Isolation of adult CMs was performed 2 days post MI andmodRNA administration. Anti-hCD25 magnetic beads were used to isolateCD25-positive cells. B CFW mice were transfected with ihCD25 and nGFPcarrying the k motif. All positive cells isolated with this approach areGFP⁺ ihCD25+. C When transfected together with L7AE carrying recognitionelements of miR1 and miR208 (CM specific modRNA) results in mixture oftransfected CMs (nGFP+ and ihCD25+) and non-transfected CMs and no-cms.D Using hCD25 magnetic beads allows one to isolate only transfected CMs.Mice=3.

FIG. 24 shows that Lin28 or Pkm2 CMs specific modRNA improve cardiacfunction 28 days post MI and injection in mouse myocardial infractionmodel. Heart function was measured for different treated groups at day 2and day 28 post MI using echocardiography. Results show improvement ofcardiac function in Lin28 or Pkm2 treated groups with synergistic effectwhen Lin28 or Pkm2 were delivered in CMs specific manner (+L7AemiR1+miR208). n=3 mice, ***, P<0.001, two-tailed student t-test.

DETAILED DESCRIPTION OF THE DISCLOSURE

All patents, published applications and other references cited hereinare hereby incorporated by reference into the present application.Methodologies used in developing the present invention are well known tothose of skill in the art unless otherwise indicated.

In the description that follows, certain conventions will be followed asregards the usage of terminology. In general, terms used herein areintended to be interpreted consistently with the meaning of those termsas they are known to those of skill in the art. Some definitions areprovide purely for the convenience of the reader.

The term “recognition element for miRNA” or “miRNA recognition elementrefers to single-stranded RNA-based oligonucleotides that are designedto bind endogenous miRNA and inhibit the expression of a constructcontaining the recognition element when it is introduced into cells.

The term “miRNA” refers to sequences that are complementary to mRNA thatare involved in the cleavage of RNA or the suppression of thetranslation. Endogenous mature miRNAs function as part of theRNA-induced complex, which has the capacity to post-transcriptionallyregulate mRNAs that have sequences with partial complementarity to thebound miRNA. Through the hybridization of the anti-miRNA sequence to themiRNA sequence, the function of the miRNA sequence is neutralized bypreventing its selective binding to the target.

The term “modRNA” refers to a synthetic modified RNA that can be usedfor expression of a gene of interest. Chemical modifications made in themodRNA, for example substitution of pseudouridine for uridine, stabilizethe molecule and enhance transcription. Additionally, unlike delivery ofprotein agents directly to a cell, which can activate the immune system,the delivery of modRNA can be achieved without immune impact. The use ofmodRNA for in vivo and in vitro expression is described in more detailin for example, WO 2012/138453.

The term “inactive human CD25” (ihCD25) refers to a truncatedinterleukin-2 receptor that has only the extracellular domain and isunable to signal into the cell. Other species, for example, inactivemouse CD25 may also be used in the disclosed method.

The present disclosure relates to methodology for achievingcell-specific expression of a modRNA encoding a gene of interest (GOI)the expression of which is desired in a cell of interest. In one aspect,the disclosure describes an expression regulatory system forcell-specific transcription, the system comprising a first nucleic acidhaving a 5′ untranslated region (UTR) and a 3′ UTR, where the nucleicacid encodes (1) a cell-specific microRNA (miR) recognition elementupstream of its 3′UTR, and (2) a translation suppressor protein; and asecond nucleic acid having a 5′ UTR and a 3′ UTR that encodes (1) asuppressor protein interaction motif, for example a K-motif, downstreamof its 5′UTR that binds the translation suppressor protein, and (2) agene that encodes a protein of interest.

Current treatments for MI address the consequences of myocyte loss, butare not effective in enhancing myocardial repair of lost heart muscle(3, 5). Recently, it was demonstrated that one day adult mammalian heartcells (mice) can regenerate heart themselves via CMs proliferation (7).Examining the genetic differences between the regenerative and thenon-regenerative stages it was found that the most differentiallyexpressed gene between these stages belong to mitosis and cell cyclecategories (7).

Modified mRNA (modRNA) has emerged as an effective and safe tool forsomatic gene transfer, and has been successfully used by us and othersfor gene delivery to the heart.^(10,12-15) Here we show that PyruvateKinase Muscle Isozyme M2 (Pkm2), a pro-proliferative factor, frequentlydysregulated in cancer,^(16,17) is highly expressed in regenerativefetal and early neonatal CMs, but not in adult CMs. Restoration of Pkm2levels using the modRNA delivery of the disclosure exclusively intoadult CMs (_(cms)Pkm2) post-MI significantly and exclusively induced CMsproliferation, and was associated with improved cardiac function,reduced scar size, increased heart to body weight ratio, reduced CMssize, reduced apoptosis and increased capillary density. Thoseregenerative processes translated into increased long-term survivalpost-MI. Using lineage tracing and isolation of Pkm2-transfected CMsfollowed by gene expression analysis post-MI we show an increase innumber of Pkm2-transfected CMs colonies and the potential involvement ofkey downstream effectors of the pro-proliferative cytoplasmic (via thepentose phosphate pathway (PPP)^(18,19)) and nuclear (viatrans-activation of β-catenin and Hif1α^(20,21)) functions of Pkm2. Ourresults show that a short pulse of a pro-proliferative gene, using ahighly translatable, clinically adaptable platform is sufficient toinduce CM proliferation and cardiac regeneration. Those findingsunderline the therapeutic potential of _(cms)Pkm2 modRNA in cardiacdisease.

Reactivation of CMs proliferation has been a key element in cardiacregeneration strategies. Zebrafish and newt cardiac regeneration ismostly mediated by CMs proliferation^(3,5,7,8). In mammals fetaldevelopment, CMs proliferation is a distinct pathway for heart growthand regeneration^(9,22.) It has been shown that after injury adult CMsupregulate a subset of fetal genes suggesting that adult CM are notterminally differentiated and possess some degree of cellplasticity^(4,9). Adult mammalian CMs can divide in vitro and in vivoand this ability can be stimulated by upregulating pro-proliferativegenes^(9,22-33). Over the years, several publications have shown thatreactivation of adult CMs cell cycle re-entry is possible viaproteins^(23,24,26,30,34) viruses ^(26,39,31,35) or transgenic mousemodels of pro-proliferation genes^(25,28,33) Protein administration forthe purpose of cell cycle induction is challenging due to the very shorthalf-life, the difficulty of local administration, lack of CMsspecificity and the inability to deliver intracellular genes, such astranscription factors. The cardiac specific adeno-associated virus(_(cms)AAV) vector is not immunogenic and used in many heart studies buthas a very long and sustained expression time that may lead to increaseduncontrolled CMs size and cardiac hypertrophy and arrhythmia. Althoughtransgenic mice can be used in CM-specific and transient way, they arenot clinically-relevant for gene delivery. Challenges with currentapproaches highlight the need for an efficient gene delivery approachthat can safely, and locally deliver cell cycle inducer genes to theCMs, with a transient, efficient, and controlled manner. Pyruvate KinaseMuscle Isozyme M2 (Pkm2) is a cell cycle inducer. During development,Pkm2 is expressed in many adult tissues including the spleen and lung,however during adulthood Pkm2 is strictly expressed in proliferatingcells with high anabolic activity^(16,17.) Pkm2 was found to increaseadult cell and cancer cells proliferation, angiogenesis and preventapoptosis caused by oxidative stress^(18,20,36-42). Pkm2 exerts itsfunctions by its two distinct functions: In the cytoplasm, Pkm2 shiftsthe metabolic fate from glycolysis to pentose phosphate pathway (PPP) byreducing the conversion of phosphoenolpyruvate to pyruvate^(18,19). Thisleads to the accumulation of galactose, a glycolysis intermediate, andactivation of PPP via Glucose-6-phosphate dehydrogenase (G6pd)⁴³⁻⁴⁵. ThePPP pathway activation leads to the synthesis of nucleotides, aminoacids, and lipids and the production of reduced NADPH, increase nitricoxide synthase and DNA repair^(38,39,41,46-48). In addition, Pkm2 has arole also in the nucleus. Pkm2 directly interacting with thetranscription factors β-catenin and Hif1α. This interaction promotes theexpression of genes such as in Ccdn1, c-Myc and Vegfa, and Bcl2^(20,21)See summary of Pkm2 role in proliferative or cancer cells in FIG. 5(S1).

Several studies indicate that cell cycle inducer genes can induce CMs toproliferate (8-22). However, activation of these genes for long periodsin CMs may lead to CMs hypertrophy and in some cases to hypertrophiccardiomyopathy and HF(14). In addition, systemic delivery of cell cycleinducer genes can lead to uncontrolled cell growth of non-CM cells inthe heart and throughout the body, and can raise safety issues.

The differential expression of different cell cycle inducer genes in theheart changes during heart development. Others and we focused on twodifferent time points after birth (Day 1 and Day 10) as they representdevelopmental stages that the heart has regenerative ability via CMsproliferation (day 1) and lacking this ability (day 10). As can be seenin FIG. 1a several cell cycle inducer changes significantly between thetwo stages of development. However Pkm2 levels in mice hearts are highduring fetal development 49 and are very significantly decreased by day10 after birth. As Pkm2 most highly significant is upstream to severalcell cycle inducer genes and his changes is the to Co-immunostaining ofPkm2 and the CMs marker α-Actinin revealed that Pkm2 was highlyexpressed in CMs during development and at one-day post birth, however,its expression was undetectable 8 weeks after birth (FIG. 1b &c). Pkm2expression in the heart post-MI was restricted to immune cells (CD45+)and non-CMs but not upregulated in CMs (FIG. 11). We have restored Pkm2levels by direct injection of Pkm2 modRNA into the myocardium (FIG. 1c). Pharmacokinetics study of Pkm2 levels after myocardial injectionindicated that Pkm2 protein expression occurred a few hours postinjection, and lasted for at least 8 days, but no longer than 12 days(FIG. 1d ). To test the effect Pkm2 expression on CMs proliferation weisolated 4-day old neonatal rat CMs and transfected them with Luccontrol or Pkm2 modRNAs (FIGS. 12A-12C). Pkm2 modRNA was translated 12hours post transfection and levels remained up to 10 days posttransfection (FIGS. 12A-12C). Three days post transfection with Pkm2 orLuc modRNAs there was a significant increase in proliferation ofPkm2-transfected CMs (FIGS. 12A-12C). To test Pkm2 effects in MIsetting, we directly injected Pkm2 or Luc modRNAs into the myocardiumimmediately after LAD ligation.13-15 One week post-MI and injection,Pkm2 significantly induced proliferation of CMs and non-CMs (FIG. 1e-h). We hypothesized that the observed improvement in proliferativecapacity may translate into better regeneration, and result in improvedoutcome post-MI. However, inducing non-CMs proliferation in the heartfrequently results in undesired effects, mainly by promoting fibrosisand immune response. Hence, we developed a unique CM-specific modRNA(cmsmodRNA) system that is based on two distinct modRNAs (FIG. 2, FIGS.13A-13C and FIGS. 14A-14C). The first construct contains L7AE, anarchaeal ribosomal protein that regulates the translation of genescontaining a kink-turn motif (K-motif), a specific binding site forL7AE.50,51 Translation of L7AE modRNA suppresses the translation of thedesigned gene of interest modRNA when the two are co-transfected intothe cell. By adding a CM-specific microRNA (cmsmiR) recognition elementto the L7AE gene 3′UTR, we were able to prevent L7AE translation in CMsthat abundantly and mostly exclusively express those miRs, allowing thetranslation of the gene of interest modRNA strictly in CMs (FIGS.13A-13C). miR1-1 (miR-1), miR-208a (miR-208) and miR-199a (miR-199) arereported to be expressed mostly in CMs.52-54 We tested the expression ofthose miRs in our model by generating an inactive human CD25 (ihCD25)—atruncated gene containing only the extracellular domain of hCD25− as areporter gene that can be immunostained when expressed on the surface ofcells/tissues. We have designed two versions of the ihCD25 construct,with or without recognition elements for miR-1, miR-208 or miR-199.modRNAs were transfected into neonatal CMs (FIGS. 14A-14C), or injectedusing the MI model (FIG. 2 ga-c and FIGS. 14A-14C). miR-1 and miR-208were found to be CM-specific, as indicated by positive ihCD25 stainingin non-CM but not in CMs. We designed a L7AE modRNA that contains bothmiR-1 and miR-208 recognition elements (miR-1-208) (FIG. 2d-k ), andused a nuclear GFP modRNA (nGFP-K) and a Cre recombinase (Cre-K) modRNAsthat contains a K-motif. In our MI model, transfection of nGFP-Kresulted in the translation of nGFP in both CMs and non-CMs. However,when nGFP-K was co-transfected with miR-1-208, nGFP was exclusivelytranslated in CMs (FIG. 2e &f). Co-transfection Cre-K with miR-1-208 inour MI model using Rosa26 reporter mice (Rosa26mTmG) resulted in GFPexpression strictly in CMs (FIG. 2g ). Injection of Cre-K alone resultedin transfection efficiency of ˜24.8% of heart section/˜2600 cells inleft ventricle (both CMs and non-CMs). However, Cre-K+miR1-208combination resulted in transfection efficiency of 7.7%/˜800 cells ofexclusively CMs (FIG. 2h ). We also show that the non-mammalian proteinL7AE does not exacerbate immune response post MI (FIGS. 15A-15C). Wehypothesize that this is due to the already active immune response inthe heart immediately post MI. We concluded that the use of L7AE in micemodel is immunologically safe. To test the functionality of ourcmsmodRNA delivery platform in our MI model, we directly injected Luc-K,miR1-208, Luc K+miR1-208, Pkm2-K, Pkm2+miR1-208 or Pkm2-K+miR1-208(cmsPkm2). Seven days post transfection we measured the proliferationrate in the heart (FIG. 2i ). Pkm2-K modRNA alone or Pkm2+miR1-208significantly increased proliferation of both CMs and non-CMs (P<0.001)compared to Luc modRNA (FIG. 2 j&k). However, cmsPkm2 modRNAsignificantly reactivated the proliferation of only CMs (P<0.001), withno significant influence on the proliferation of non-CMs. Using liveimaging of neonatal rat CMs for 24 hours, we found that co-transfectionof cmsPkm2 modRNA with cmsnGFP modRNA increased CMs proliferation incomparison to transfection with cmsnGFP modRNA alone (Supplemental Movie1). Additionally, cmsPkm2 modRNA significantly reduced apoptosis andincreased capillary density in the myocardium 2 or 7 days post-MI (FIGS.16A-16C). MRI or echo showed that cmsPkm2 significantly increased thepercentage of ejection fraction (FIG. 3a-d and Supplemental Movies 2&3)and delta of percentage fractioning shortening from day 2 (baseline) today 28 post-MI (FIG. 3d ). Left ventricular internal diameter endsystole was increased, while left ventricular internal diameter enddiastole was significantly increased in cmsPkm2 mice compared to control28 days post-MI (FIGS. 16A-6C). 28 days post-MI, Pkm2 or cmsPkm2expression significantly reduced cardiac scar formation Additionally, noabnormality in the cardiac tissue (e.g. angioma, edema) was observedpost injection of cmsPkm2 (FIG. 3e &f), heart weight to body weightratio was significantly increased (FIG. 3g ) while CMs size wassignificantly decreased, indicating the CMs proliferation (FIG. 3h andFIGS. 16A-16C), and capillary density was significantly increased (FIG.3i ) in Pkm2 or cmsPkm2 modRNA transfections compared to controls.Lastly, cmsPkm2 significantly increased CMs number in the heart withoutelevating the number of nuclei per CM, while increasing the mononuclearfraction compared to control (FIG. 3j-m ). Importantly, long-termsurvival curve for mice treated immediately after MI with cmsLuc orcmsPkm2 modRNAs showed significant improvement in mice survival post-MIand cmsPkm2 transfection (FIG. 3n ). To understand the mechanism bywhich cmsPkm2 improves cardiac function post-MI, we used alineage-tracing model that combines cmsmodRNAs and R26mTmG (FIG. 4a-j )to exclusively express Pkm2/Luc in CMs (by mixing cmsPkm2/cmsLuc+cmsCremodRNA; GFP-labeled CMs, FIG. 4a &b), and trace the fate and propertiesof transfected CMs over time, after the cmsmodRNA was no longerexpressed. The number of CMs transfected with cmsPkm2+Cre modRNAs washigher 3 days post-MI and significantly higher 28 days post-MI comparedto control (FIG. 4c &d). Heart weight to body weight ratio wassignificantly increased (FIG. 4e ), while GFP⁺ CMs size (FIG. 4f ) andnuclei number/cell (FIG. 4g ) was significantly decreased in micetreated with cmsPkm2+Cre modRNA compared to control. Importantly, 28days post treatment with cmsPkm2+Cre modRNA, GFP⁺ CMs showed elevatedexpression of proliferative markers such as pH3 and Ki67 (FIG. 4h-j ),long after Pkm2 was not expressed. Changes in gene expression postcmsPkm2 or cmsLuc together with cmsihCD25 modRNAs delivery in MI settingwere measured 2 days post injection. Isolated cells were enriched forCMs markers with significantly lower expression of Troponin T (FIG. 4k). Pkm2 expressing cells significantly upregulated effectors downstreamof its cytoplasmic (G6pd) and nuclear (c-Myc, Cyclin D1, Bcl2, VEGF-Aand Pdk1) functions (FIG. 4l ). In accordance with the increasedproliferation, we observed an upregulation of cell cycle-promoting genes(Cdc20, Cdk1 and Ccnd2, Ccnb1), and downregulation of the cell cycleinhibitors (p21 and p27) in Pkm2+ ihCD25+ CMs compared Luc+ ihCD25+ CMs(FIG. 4M).

The rapid downregulation of Pkm2 after birth, which coincides with theloss of cardiac regeneration ability,⁵⁵ points to its involvement infetal and neonatal cardiac regeneration. Additionally, itspreviously-described pro-proliferative and pro-survival roles in cancer,make it an ideal candidate to promote cardiac function/regeneration. Ourfinding that _(cms)Pkm2 improves outcome after MI, most likely byimproving cardiac function, has physiological and clinical implications,as they underline the potential therapeutic value of _(cms)Pkm2expression immediately post-MI. Our results are in agreement with arecent publication showing that a short expression of synthetic miRs issufficient for the induction of CMs proliferation and cardiacregeneration⁵⁶. In addition, the cardiac specificity of our modRNA alongwith its short expression time make it a safe and translatable strategyfor cardiac regeneration. Our data point to the high potency of Pkm2 andits ability to induce metabolic reprogramming that better supports CMshomeostasis with long-term beneficial effects, lasting weeks after theprotein was no longer expressed. Our experimental approach and toolswill allow us to further investigate other relevant pro-proliferativeand metabolic reprogramming genes and their therapeutic potential indifferent disease models, and to efficiently and precisely study CMscell fate. Notably, our isolation approach using _(cms)ihCD25 (FIGS.17A-17C) overcomes the challenge of FACS sorting of adult transfectedCMs.³¹ This study pioneers the use of _(cms)modRNAs to manipulatecellular behavior and holds a great therapeutic potential for cardiacdisease, as modRNA is a safe, transient, local, and non-immunogenicplatform for gene transfer.

The field of cardiac gene therapy is expanding, yet its use in theclinical setting is limited. Currently the most widely used method fortargeting gene expression to the heart is through viral vectors,particularly the adeno-associated virus (AAV) vector (1-3). During thepast few decades several attempts were made to insert genes of interestinto CMs using adenovirus, associate adeno virus (AAV), lentivirus andDNA plasmid. While both AAV and adenovirus possess high CM transfectionlevels, lentivirus and DNA plasmid CMs transfection efficiency is low.Adenoviruses can elicit a robust immune response, leaving only AAV as asuitable option for gene delivery system to the heart. UsingCMs-specific promoters in AAV may allow for cell-cycle inducers geneexpression strictly in CMs, however its pharmacokinetics in the heart(expression starts at day 4 and remains for at least 11 months) may leadto uncontrolled growth and hypertrophic cardiomyopathy and HF (3, 5).Additionally, over 60% of healthy human individuals possess neutralizingantibodies directed against the AAV capsid that can efficientlyneutralize gene expression delivered by this method (21). Viral genetherapy shows promise yet its applications are limited due to its lengthof expression and inability to regulate gene expression in aquantifiable dose manner (1-3).

While the use of unmodified exogenous RNA as a gene delivery method isappealing because it may be safer than plasmid DNA owing to a reducedrisk of genomic integration, it is ineffective due to its instabilityoutside the cell and the strong innate immune response it elicits whentransfected into cells (10,11).

Kariko et al. discovered that the substitution of Uridine and Cytidinewith Pseudouridine and 5-methylcytidine, respectively, drasticallyreduced the immune response elicited from exogenous RNA (11,12). Inorder to increase stability and translational efficiency, a3″-O-Me-m7G(5′)ppp(5′)G Anti Reverse Cap Analog (ARCA) cap issubstituted at the 5′ end of the RNA molecule (4,5,10). Modified mRNA(modRNA) therefore provides a novel and effective gene delivery methodthat provides short-term (1-2 weeks), titratable gene expression for useboth in vitro or in vivo (4-9).

Modified mRNA (modRNA) has emerged as an effective and safe tool forsomatic gene transfer, and has been successfully used by us and othersfor gene delivery to the heart.^(10,12-15) Here we show that PyruvateKinase Muscle Isozyme M2 (Pkm2), a pro-proliferative factor, frequentlydysregulated in cancer,^(16,17) is highly expressed in regenerativefetal and early neonatal CMs, but not in adult CMs. Restoration of Pkm2levels using modRNA delivery exclusively into adult CMs (_(cms)Pkm2)post-MI significantly and exclusively induced CMs proliferation, and wasassociated with improved cardiac function, reduced scar size, increasedheart to body weight ratio, reduced CMs size, reduced apoptosis andincreased capillary density. Those regenerative processes translatedinto increased long-term survival post-MI. Using lineage tracing andisolation of Pkm2-transfected CMs followed by gene expression analysispost-MI we show an increase in number of Pkm2-transfected CMs coloniesand the potential involvement of key downstream effectors of thepro-proliferative cytoplasmic (via the pentose phosphate pathway (PPP)18,19) and nuclear (via trans-activation of β-catenin and Hif1α^(20,21))functions of Pkm2. Our results show that a short pulse of apro-proliferative gene, using a highly translatable, clinicallyadaptable platform is sufficient to induce CM proliferation and cardiacregeneration. Those findings underline the therapeutic potential of_(cms)Pkm2 modRNA in cardiac disease.

It has recently been shown (1) that by using modified mRNA (modRNA)technology, modRNA can drive a transient, safe gene expression in theheart with high transfection levels without eliciting immune response orcompromising the genome(5, 22). Exogenous unmodified mRNA that entersthe cell via the cell membrane is recognized by endosomal Toll-likereceptors 7/8 and 3(23, 24). This process inhibits protein translationand activates the innate immune response, ultimately leading toapoptosis of the hosting cell. ModRNA is synthesized by substitutingribonucleotides with naturally modified ribonucleotides. The use ofthese modified ribonucleotides results in changing the secondarystructure of the synthesized mRNA, which prevents the Toll-likereceptors from recognizing the modRNA and therefore permitting itstranslation to a functional protein by the ribosomal machinery withouteliciting immune response or compromising the genome (5, 22).

Applicants previously showed that modRNA transfects different cell typesin the heart including CMs with high efficiency, leading to immediateand high levels of protein expression in a transient, pulse like kinetic(duration of 3-5 days in vitro and 7-10 days in vivo). Co-transfectionof two individual modRNAs resulted in co-translation of both. Using theMI model (5) and Luc, LacZ and nGFP modRNAs delivery in myocardium,Applicants show that the cardiac tissue after MI is well transfectedwith modRNA and several cell types such as CMs and non-CMs are highlytransfected in the left ventricle. Applicants then selected severalcandidate cell cycle inducer genes that had previously been shown tohave the ability to induce neonatal CMs during cardiac development(CDK2, β catenin) (16) or reactivation of adult CMs proliferation intransgenic mouse models (CyclinD2, cMYC)(12, 14) and others that hadshown robust proliferative potential in different organs and cell typesbut had never been tested in cardiomyocytes and heart (Lin28, PKM2)(24,25).

Generally, a platform for making cell specific modified mRNA (modRNA) isas follows.

First, choose a cell type of interest for making cell specific modRNA.Identify candidate microRNA (miR) that have been reported to express inthe cell of interest and preferably only in the cell of interest (e.g.,in the case of cardiomyocytes, miR1, miR29, miR126, miR133, miR199,miR208, miR378). Identify reverse complement sequences for each miRsequence that allows recognition of the specific miR to this sequence.Add to 3′UTR each of the previous calculated miR reverse complementsequence to ihCD25 k motif, a truncated receptor for hCD25 carrying a kmotif. This allows ihCD25 to express only in those cells that arelacking the specific miR that the reverse complement sequence istargeting.

Co-transfect a mixture of cells that contains the cell of interest andother cell type (e.g fibroblasts) as well with nGFP modRNA and withdifferent miR-ihCD25 modRNAs. After about 18 hours, fix the cells andstain the cells for GFP (show transfected cells with modRNA) and forreporter gene (with anti hCD25, show cells that are lacking the miR thatwas target) and cell specific markers (e.g., for cardiomyocytes TroponinI, for endothelial cells, Pecam1, etc.).

Identify GFP-positive cells that are also positive for cell specificmarker (e.g Troponin I for cardiomyocytes) but negative for reportergene (hCD25). This means that this specific miR-ihCD25 was nottranslated although the modRNA was delivered to this cell type. Thiswill indicate that this miR is specifically expressed in the cell typeof interest and can be used to create cell specific modRNA. Create cellspecific modRNA by adding to the 3′UTR of L7AE the sequence thatinhibits ihCD25 in the cell of interest. Co-transfect with mir-L7AE andgene of interest that carrying in his 5′ UTR k-motif. These two modRNAswill allow you to specifically deliver a gene of interest to a specificcell type.

In one embodiment, Applicant designed and generated modRNAs for each ofthe above genes. Using rat neonatal CMs, Applicant tested thetranslation of each modRNA. In addition, the functionality of theprotein was tested by measuring the proliferation rate of rat neonatalCMs with control and the candidate cell inducer modRNAs. All candidatecell cycle inducer modRNAs increase the proliferation of neonatal ratCMs and adult CMs proliferation after MI to various extents. Both Lin28and PKM2 significantly increased CMs proliferative capacity. Therefore,those genes were selected for further investigation.

Lin28 is a known suppressor of Let7 that tightly controls cell cycleregulators (25-29). To test whether Lin28 induces cell cycle regulators,nGFP (control modRNA) or Lin28 modRNA was injected immediately after LADligation and found a significant increase in the expression of Ccnb1,Ccnb2, Cdc20, Cdk1 and Aurka cell cycle genes after 3 days using RT-PCR.The use of cell cycle inducer modRNAs such as Lin28 modRNA in anon-specific manner increases proliferation not only in CMs, but alsonon-CMs representing an experiential challenge since the model andhypothesis were aimed to test aimed to test CMs proliferation as a meanto achieve increased cardiac regeneration.

To address this challenge Applicants designed a CM-specific modRNAsystem that is based on two distinct modRNAs (FIG. 5). The firstconstruct is a suppressor modRNA the carries L7AE, an archaeal ribosomalprotein that regulates the translation of a designed gene of interestmodRNA with kink-turn motif—a specific binding site for L7AE(30, 31).Translation of L7AE modRNA will suppress the translation of the designedgene of interest modRNA when the two are co-transfected into the cell.By adding a CMs-specific microRNA (miR) recognition element to the L7AEgene, we are able to prevent L7AE translation in CMs that abundantly andmostly exclusively express the miR (“suppress the suppressor” approach)allowing the translation of the gene of interest modRNA strictly in CMs.It was shown previously, using miR recognition element, results in areduction of the number of copies of the targeted miR (32,33). Reductionin number of miRs in the heart can be detrimental or beneficial to theheart (33-42). In our approach we need to be sure we don't reduce miRexpression that is beneficial to cardiac regeneration but ratherreducing miR expression that is detrimental to cardiac regeneration.miR1-2 (miR1), miR208a (miR208) and miR199a (miR199) are expressedmostly in CMs (33, 39, 41). miR1 and miR208 were found to be upregulateafter MI in adult animal study and humans (33, 38, 41, 43). miR1 andmiR208 up regulation has detrimental effects, while its down regulationhas beneficial effects after MI and heart diseases (32-42)

To test the expression of these miRs in CMs, we have made an inactivehuman CD25 (ihCD25) gene, a truncated gene containing only theextracellular domain (ECD) of hCD25− as a reporter gene. We havedesigned two versions of the ihCD25 construct, with or without the miRrecognition elements for miR-1, miR-208 or miR-199. We then transfectedthe modRNAs into neonatal CMs in vitro and in vivo using the MI model(FIG. 2). As can be seen in FIG. 6 both miR-1 and miR-208 were found tobe CM-specific, as translation of ihCD25 was observed in non-CM but notin CMs. In contrast, modRNAs with or without miR-199 recognition elementwas found not to be CMs specific, in vitro and in vivo. Next, wedesigned a L7AE modRNA that carries both miR-1 and miR-208 recognitionelements (L7AE miR-1+miR-208). We have also generated a nuclear GFPmodRNA (nGFP-k-motif) and a destabilized Cre recombinase (DD-Cre-kmotif) modRNAs that includes the k-motif (L7AE recognition site). Usingour adult mouse MI model, we show that transfection of nGFP-k motifresulted in the translation of nGFP in both CMs and non-CMs (FIG. 7).However, when nGFP-k motif was co-transfected with L7AE miR-1+miR-208only CMs translated the nGFP. In addition, co-transfecting L7AEmiR-1+miR-208 with a DD-Cre-k motif in a MI model usingRosa26^(Tdtomato) resulted in gene activation (Tomato fluorescence)strictly in CMs. The combination of these two methods allows us toelegantly express our gene/genes of interest exclusively in CMs, and toallow for linage tracing over longer time periods after the gene ofinterest modRNA is no longer expressed.

To test the functionality of our CMs-specific modRNA, we directly injectLuc control modRNA or Lin28-K and PKM2-k motif modRNA alone or togetherwith L7AE miR-1+miR-208 (Lin28/PKM2 CMs specific modRNA) using our MImodel. Seven days post transfection we measured the proliferation (usinghallmark proliferation markers, BrdU, Ki67, H3P and Aurora B) of bothCMs and non-CMs. As depicted in FIG. 8 Lin28-k or PKM2-k motif modRNAalone significantly increased proliferation of both CMs and non-CMs(P<0.001) in comparison to Luc modRNA. However, Lin28 and PKM2CMs-specific modRNA significantly reactivated the proliferation of onlyCMs (P<0.001), with no significant influence on the proliferation ofnon-CMs. Importantly, since L7AE is not a mammalian protein, to test theimmunogenicity of L7AE after MI we have injected Luc control modRNA orL7AE modRNA with or without miR-1, miR-208 or both in adult mouse MImodel. As can be seen in FIG. 8 we did not witness significantelevations in immune response and increased apoptosis with all L7AEmodRNAs after 7 day post MI. We concluded that the use of L7AE in miceis immunologically safe.

Plasmids

pTEMPLZ is a cloning vector into which an ORF of interest can beinserted between the UTRs. In one embodiment, plasmids for use in thedisclosed method include those shown in Table 1.

TABLE 1 1 No miR-L7AE

2 miR 1-L7AE

3 miR 208a-L7AE

4 miR 1-miR 208a-L7AE

5 Lin28-K motif

6 Pkm2-K motif

7 nucGFP-K motif

8 ihCD25 K motif-non modified

ihCD25 modRNA-Based CM-Specific Cell Sorting

To test whether our novel CMs-specific modRNA-based design that allows atransient gene expression of our genes of interest exclusively in CMscan be used to isolate only transfected CMs with innovative inactive(only extracellular domain) human CD25 (ihCD25)-based sorting (magneticbeads) system we injected nGFP k-motif with ihCD25 k-motif modRNA inheart after MI, cells were Isolated and sorted out with CD25-specificmagnetic beads. Both nGFP-positive CM's and non-CM's were observed. WhennGFP k-motif, ihCD25 k-motif with L7AE miR1-miR208 were co-transfectedand without magnetic separation, CM specific nGFP expression was seen.The culture also contains nGFP-negative CMs and non-CMs. When magneticseparation was applied, only pure nGFP-positive CMs were observed (FIG.9).

In one embodiment, production of exogenous genes is driven by expressionof anti-miRs from a first replicon that also encodes a repressorprotein. Expressed anti-miRs bind miRs that occur naturally in human andprimate cardiomyocytes and transcription of the repressor protein isprevented. In the absence of repressor protein, expression of a gene ofinterest from a second replicon encoding the gene and containing therepressor protein recognition site can proceed.

Cell Cycle Inducer Genes

Expression of a gene of interest, for example, a proliferation-inducinggene can be made cardiomyocyte-specific by placingtranscription/translation of the gene under the control of atranscription/translational regulatory system in which one of a pair ofnucleic acids encodes an anti-microRNA (anti-miR) that bindsspecifically to a target cardiomyocyte-specific miR. A second nucleicacid translation suppressor protein and a second nucleic acid thatcomprises a suppressor protein interaction motif that binds thetranslation suppressor protein and a gene that encodes a protein ofinterest.

Using the method described herein, the expression is transient, avoidingthe problems associated with unlimited expression, such as hypertrophy.

Repressor/suppressor protein that binds a specific RNA motif inserted inthe 5′-untranslated region of an mRNA modulates the translation of thatmessage in mammalian cells. The expression specificity to human andprimate cardiomyocytes is achieved by the inclusion in therepressor/suppressor oligonucleotide of a sequence that encodes arecognition element specific to endogenous miRNAs found in mouse, pig,human and non-human primate cardiomyocytes.

The synthesis of modRNA for in vivo use involves four stages: DNAtemplate creation containing the desired transcript, in vitrotranscription (IVT), 5′ phosphate removal with Antarctic phosphatase,and precipitation with 5M ammonium acetate salt. Investigation into theuse of modRNA for experimental and clinical purposes is growing rapidly.Daily transfection with modRNA encoding reprograming factors OCT4, SOX2,MYC, and KLF4 were successful at reprogramming human fibroblasts back topluripotency (5,8). Additionally, modRNA has been shown to be capable ofdirecting cell fate in vitro by using MyoD modRNA that resulted in theconversion of fibroblasts to skeletal muscle cells (2). ModRNA has alsoshown promise in directing cell fate in vivo. The expanding use ofmodRNA technology in vivo and its potential use in the field of cardiacgene therapy motivated us to generate a step-wise, streamlined protocolfor the effective synthesis of modRNA for in vivo use.

Cardiomyocytes

In one embodiment, the present disclosure relates to a method oftreating a subject following myocardial infarction (MI) or heart failure(HF) in a subject comprising administering an effective amount of acomposition comprising at least two synthetic modRNAs to a subject inneed thereof.

Protein expression lasts for from 5 to 20 days, in some embodiments from7 to 14 days, and results in a low immunological response as compared tonon-modified RNA.

Inter alia, the present disclosure describes a new set of candidate cellcycle inducer genes: Lin28, Pkm2, and Cyclin D2, which when delivered asmodRNA, can reactivate mammalian cardiomyocyte (CM) proliferation invivo (without increasing CM size or nuclei number), reduce CM apoptosisand increase overall left ventricle vascularization post myocardialinfarction (MI). When expression of the cell cycle inducer genes isplaced under the control of a transcriptional/translational regulatory(an expression regulatory) system for cardiomyocyte-specifictranscription (expression), the result is a tool for cardiomyocytespecific expression of the cell cycle inducer gene-driven proliferationfollowing injury, for example, as the result of myocardial infarction(MI) or heart failure (HF).

Modified mRNA (modRNA) is a safe, efficient, transient, andnon-immunogenic gene delivery system that allows one to investigate theeffect of cell cycle inducer genes on CMs following MI or HF. Kariko etal. discovered that the substitution of uridine and cytidine withpseudouridine and 5-methylcytidine, respectively, drastically reducedthe immune response elicited from exogenous RNA (11,12). Investigationinto the mechanism revealed that the nucleoside substitutions resultedin a conformational change in the RNA that caused reduced response bytoll-like receptors 3, 7, and 8 (TLR3, TLR7, TLR 8), and retinoicacid-inducible gene 1 (RIG-1) (13). A further decrease in RIG-1 responsefrom modRNA was seen upon removal of the 5′ triphosphates (4,10). Inorder to increase stability and translational efficiency, a3″-O-Me-m7G(5′)ppp(5′)G Anti Reverse Cap Analog (ARCA) cap issubstituted at the 5′ end of the RNA molecule (4,5,10).

Cell Selection by Anti-hCD25 Affinity

Cell selection of cardiomyocytes by traditional FACS cell sorting can beproblematic due to the size of the cells. An alternative approach tocell selection was devised. In one embodiment, using the transcriptionregulatory system of the disclosure, a nucleic acid that encodes thehCD25 extracellular domain (ECD) is included in the construct thatcontains the nucleic acid that encodes the gene of interest. To isolateCMs that transiently express either control or candidate gene modRNA,cells that co-express the hCD25 ECD plus the gene of interest areselected using an anti-CD25 ECD antibody in an affinity chromatographycolumn or using a panning method. These cells are used to generate geneexpression profiles using RNA-seq technique, and identify differentiallyexpressed genes

EXAMPLES Example 1: Materials

The following materials are used in conjunction with the disclosedmethod.

All solutions should be made in Nuclease Free water unless otherwisespecified. All materials used in this protocol should be nuclease free.

Equipment used includes the following:

-   -   1. PCR thermocycler    -   2. Microfuge    -   3. Vortex mixer    -   4. Thermomixer (EPPENDORF™)    -   5. Nano-Drop    -   6. Nuclease-free water    -   7. 15 ml Nuclease Free conical tubes    -   8. Nuclease free strip PCR tubes    -   9. Ethanol (100% and 70%)    -   10. 2 ml Ambion Elution Tubes

Primers used for tail PCR are as follows:

Forward Primer:  (SEQ ID NO: 9)5'-TTG GAC CCT CGT ACA GAA GCT AAT ACG-3'  Reverse Primer: (SEQ ID NO: 10) 5'-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT  TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT  TTT TTT TTT TTT TTT TCT TCC TAC TCA GGC TTT ATT CAA AGA CCA-3'

Construction of DNA template for in vitro transcription using pTEMPLZplasmid is as follows:

-   -   1. T4 Polynucleotide kinase enzyme    -   2. 100 mM ATP    -   3. 2×KAPA HiFi HotStart ReadyMix PCR master mix    -   4. Alel enzyme    -   5. Afel enzyme    -   6. Antarctic phosphatase enzyme    -   7. T4 DNA ligase enzyme    -   8. One Shot® ccdB Survival™ 2 T1 Phage-Resistant (T1R) cells    -   9. QIAquick® gel extraction kit    -   10. QIAquick® PCR purification kit    -   11. One shot chemically competent E. coli    -   12. QIAprep® spin Miniprep kit    -   13. 10× Phosphorylation Buffer

Synthesis of Linear DNA Template with a Poly T Tail for IVT Reaction

-   -   1. 2×KAPA HiFi HotStart ReadyMix PCR master mix    -   2. Primer Solution: 1 μM each of forward and reverse primer    -   3. Dpnl enzyme    -   4. QIAquick® PCR purification kit

In Vitro Transcription Reaction

-   -   1. Ambion T7 Megascript Kit (life technologies Cat #: am1334-5)    -   2. GTP 75 mM solution (provided in Megascript® kit)    -   3. ATP 75 mM solution (provided in Megascript® kit)    -   4. CTP 75 mM solution (provided in Megascript® kit)    -   5. 5-Methylpseudouridine-5′-Triphosphate (Trilink)    -   6. Trilink Biotechnologies Anti Reverse Cap Analog,        3′-O-Me-m⁷G(5′)ppp(5′)G 10 μmoles (Cat #: N-7003)    -   7. T7 TURBO DNase enzyme (provided in Megascript kit)    -   8. Ambion MEGAclear™ Transcription Clean-Up kit. (life        technologies; cat #: AM1908).

RNA Phosphatase Treatment

-   -   1. Antarctic phosphatase enzyme

RNA Precipitation

-   -   1. 5M Ammonium Acetate Salt solution (Provided in        AmbionMEGAclear™ Kit)    -   2. Elution Buffer (Provided in AmbionMEGAclear™ Kit)

Preparation for modRNA injection

-   -   1. Lipofectamine® RNAiMAXtransfection reagent (Thermofisher Cat        #: 13778150)    -   2. OptiMEM Reduced Serum Medium, no phenol red    -   3. Ultra-Fine insulin syringe needle 31 g 8 mm

Methods

The following methods are used in conjunction with the disclosed method.All procedures were carried out at room temperature, in a non-sterileenvironment unless otherwise specified. All materials used should benuclease free.

Synthesis of modRNA

ModRNAs were transcribed in vitro from a plasmid templates using acustom ribonucleotide blend of anti-reverse cap analog,3″-O-Me-m7G(5′)ppp(5′)G (6 mM, TriLink Biotechnologies), guanosinetriphosphate (1.5 mM, Life Technology), adenosine triphosphate (7.5 mM,Life Technology), cytidine triphosphate (7.5 mM, Life Technology) andN1-Methylpseudouridine-5′-Triphosphate (7.5 mM, TriLink Biotechnologies)as described previously.⁴⁸⁻⁵⁰ mRNA was purified using megaclear kit(Life Technology) and was treated with antarctic phosphatase (NewEngland Biolabs), followed by re-purification using Megaclear kit. mRNAwas quantitated by Nanodrop (Thermo Scientific), precipitated withethanol and ammonium acetate, and resuspended in 10 mM TrisHCl, 1 mMEDTA. For a detailed protocol please see our recent publication.⁴⁸

modRNA transfection. In vivo transfection of modRNA was done usingsucrose citrate buffer containing 20 μl of sucrose in nuclease-freewater (0.3 g/ml), 20 μl of citrate (0.1M pH=7; Sigma) mixed with 20 μlof different concentrations of modRNA in saline to a total volume of60p1. The transfection mixture was directly injected (3 individualinjections, 20 μl each) into the myocardium. For in vitro transfection,we used RNAiMAX transfection reagent (Life Technologies) that was usedaccording to manufacturer's recommendation.

Construction of DNA Template for In Vitro Transcription Using pTEMPLZPlasmid Carrying k Motif.

pTEMPLZ is a cloning vector into which an ORF of interest can beinserted between the UTR's (FIG. 1). The 5′- and 3′-UTRs are synthesizedde novo by synthetic oligos. The synthesized UTR's are annealed togetherand amplified using forward and reverse primers. To provide an entrysite for the ORF, Alel and Afel restriction sites are introduced inbetween 5′ and 3′ UTRs. (The adenine nucleotide (A) of the 1st codon(ATG) can be omitted from forward primer sequence as it is provided bythe Alel site.) The PCR-amplified fragment and pZErO-2 vector withampicillin resistance are digested with HindIII and NotI, and ligatedtogether to create pTEMPLZ. (pTEMPLZ plasmid and derivatives should bepropagated in bacterial strain resistant to the ccdB gene product suchas One Shot ccdB Survival™ 2 T1 Phage-Resistant (T1R) cells.)

Before insertion into pTEMPLZ, the ORF is amplified by usingphosphorylated forward and reverse primer pair for the gene of interest.Phosphorylation of the primers is done using the T4 polynucleotidekinase enzyme according to reaction below:

10 x Phosphorylation Buffer 5 μl Forward primer (100 μM) 3 μl ReversePrimer (100 μM) 3 μl 100 mM ATP 0.5 μl T4 polynucleotide kinase 10 UNuclease free Water 50 μlIncubate reaction at 37° C. for 1 hour.To inactivate enzyme, the reaction is incubated at 65° C. for 20 min.The reaction is diluted to 300 μl by adding 250 μl of water giving final1 μM primer mixture.

Amplification of the ORF of interest is done using the PCR reactionbelow:

Primer mix (1 μM) from above 10 μl Template DNA 1-100 ng Water 50 μlHiFi HotStart ready mix (2 x) 25 μlThe mixture is run in Thermocycler with settings according to FIG. 2.The amplified target is isolated using QIAquick gel extraction kit.Before insertion of ORF, pTEMPIz is linearized and dephosphorylated. Tolinearize plasmid, pTEMPIz is digested with Alel and Afel according tothe reaction below:

pTEMPLZ Plasmid DNA 2 μg Nuclease Free Water 30 μl 10 x Buffer 4 3 μlAlel 5 U Afel 5 UIncubate in thermomixer for 1 hour at 37° C. Digest was purified usingQIAquick® PCR purification kit and eluted in 30 μl of elution buffer.

Linearized pTEMPLZ is dephosphorylated according to reaction below:

Linearized μlasmid from step 1 30 μl 10 x antarctic phosphatase buffer 5μl Antarctic phosphatase 5 U Nuclease Free Water 50 μlThe reaction is incubated at 37° C. for 1 hour. Enzyme is inactivated byincubating at 65° C. for 15 min.

Linearized and dephosphorylated plasmid is isolated using QIAquick gelextraction kit and the quantity of pTEMPLZ product is determined usingnanodrop. Plasmid can be stored in −20° C. for future use.

Blunt end ligation of ORF of interest is performed into pTEMPLZaccording to the reaction below:

Linearized dephosphorylated TEMPlz μlasm id 50 ng Amplified ORF 3-foldmolar excess 10 x T4 DNA ligase buffer 2 μl T4 DNA ligase 4 U NucleaseFree Water 20 μlMix reagents and incubate overnight on melting ice at room temp or at16° C. Negative control ligation reaction might be necessary to monitorself-ligation of plasmid. Transformation of plasmid is performed withcompetent cells and grow on an ampicillin agar plate.

To isolate positive clones with the correct orientation colony PCR isperformed. Between 8-10 colonies are extracted from ampicillin agarplates with a pipette tip. Individual tips are stabbed in 200 μl ofLuria Broth (LB) and rinsed several times in 75 μl of TE buffer under pH8.0, tips are incubated in 37° C. in a shaker. Tubes are then boiled for5 min to lyse bacteria and spun to pellet debris. Colony PCR isperformed with 2 μl of supernatant using forward primer and genespecific reverse primer. PCR sample is run on 1% agarose gel to identifyclones with positive orientation. 200 μl of LB is cultured with correctorientation clones in larger volume of LB overnight in a 37° C. shakerand extracted using QIAprep® spin Miniprep kit. The quantity of plasmidproduct is determined using NANODROP™ (Thermo Fisher Scientific) anddiluted to a concentration between 1-5 ng/ul.

Synthesis of Tailed DNA Template

A 1600 μl PCR master solution was prepared according to the reactionbelow:

Plasmid solution (1-5 ng/μl) (see Note 3) 400 μl Primer solution (1 μMprimers) 400 μl 2X KAPA HiFi HotStart ReadyMix. 800 μl50 μl of PCR master solution was aliquoted into 32 separate PCR tubes.PCR is run using the thermo cycler (setting listed in FIG. 2). Thelength of elongation step may vary depending on DNA polymerase used andORF length (If using 2×KAPA HiFi HotStart ReadyMix, for example,elongation step of Thermocycler should be set at a ratio of 30 sec perKb of ORF length.).

To digest methylated plasmid DNA, product is combined into oneEPPENDORF™ tube and digested with 30 μl of Dpnl. The PCR product ispurified using QIAquick PCR Purification Kit (Qiagen cat #: 28106) andthe final product eluted in nuclease free water. The concentration oftailed product is measured using nanodrop machine and concentration isadjusted using nuclease free water to 100-200 ng/μl.

For quality control analysis, purity of Tailed DNA template product ischecked on a 1% agarose gel together with the original DNA plasmid (FIG.3a ).

In Vitro Transcription (IVT) Reaction (1 ml Reaction Volume)

A custom NTP mixture is prepared in one EPPENDORF™ tube according toTable 2. Reagents for IVT reaction are mixed in the following order intoone EPPENDORF™ tube:

-   -   a. 400 μl of custom NTP's from table 2.    -   b. 400 μl of the DNA tailed template (200 ng/μl).    -   c. Vortex 10× Reaction Buffer from the T7 megascript kit to        dissolve any precipitate and add 100 μl.    -   d. Add 100 μl of T7 Enzyme. This will give you a 1 ml IVT        reaction    -   e. Mix thoroughly and Incubate in thermomixer at 37° C. for 4-6        hours. 30 μl of T7 Turbo DNase was added and mixed gently and        then incubated at 37° C. in Thermomixer for 15-20 min to halt        the reaction (see Note 5).        Purify reaction using Ambion MEGAclear™ Transcription Clean-Up        kit and elute each tube three times with 50 μl of 95° C. elution        buffer to obtain 150 μl of RNA product in each tube. Combine the        RNA mixture from each tube into one EPPENDORF™ tube.

RNA Phosphatase Treatment

Nuclease-free water is added to the RNA to obtain a 1.5 ml solution. 150μl of Antarctic Phosphatase Buffer (10×) and 150 μl of AntarcticPhosphatase enzyme is added, mixed thoroughly and incubated inthermomixer at 37° C. for 1 hour.

RNA Precipitation Using Ammonium Acetate

The 1800 μl RNA solution is transferred to a 15 ml conical tube. 180 μlof 5M ammonium acetate is added and mixed thoroughly. 5200 μl of cold(−20° C.) 100% ethanol is added to solution and aliquoted into 3-4 2 mlEPPENDORF™ tubes. Let tubes stand in −20° C. overnight. The tubes arecentrifuged at 10,000 rpm for 30 min at 4° C. The supernatant is thencarefully discarded. Each pellet is dissolved in 500 μl of 70% ethanol.modRNA ethanol solutions from each tube are consolidated into 1EPPENDORF™ tube. The tube is centrifuged at 10,000 rpm for 30 min at 4°C. The supernatant is gently poured out and discarded, and using akimwipe, the inside of the tube is gently cleaned. Care is taken not todisturb the pellet. The tube is inverted and let stand for no more than2 min to air-dry pellet. Using a pipette, any small drops of ethanolleft around the pellet are gently removed. The pellet is resuspendedusing 45-50 μl of elution buffer. modRNA is left in elution buffer for 5min then gently pipetted until the pellet is dissolved. RNA solution cannow be used in vivo, stored in −20° C. for up to 6 months, or −80° C.for 5 years.

ModRNA Yield

Concentration is measured using nanodrop machine (FIG. 3b ). The ratioof A260/A280 should be greater than 1.8 with values closer to 2.0indicating higher purity. Depending on yield, concentration should beclose to 20 μg/ul. For better quality control analysis a 1 μl samplefrom the final modRNA solution is diluted in 100 μl of nuclease freewater. The sample is analyzed using a bioanalyzer machine (FIG. 3c ).

Preparation of modRNA for Myocardial Injection in Mice

40 μl of RNAiMax is combined with 5 μl of OptiMEM in an EPPENDORF™ tubeand vortexed. The mixture is allowed to sit for 10 min at roomtemperature. In another EPPENDORF™ tube 150-200 ug of modRNA is combinedwith 5 μl of OptiMEM. The tube is spun down to eliminate liquid on thesides of the tube. After letting the RNAiMAX and OptiMEM mixture sit for10 min at room temperature, the liquid from the tube with the modRNAmixture is added to the tube with the RNAiMAX mixture. (In someembodiments, it is important to add the modRNA mixture to the RNAiMAXmixture and not the other way around.) The combined mixture is allowedto stand for 15 min at room temperature. The mixture is extracted into a31 gauge insulin syringe and injected into mouse myocardium. (Example ofresult shown in FIG. 4).

Mice

All animal procedures were performed under protocols approved by theIcahn School of Medicine at Mount Sinai Institutional Care and UseCommittee. CFW (Swiss Webster) mice or Rosa26^(mTmG) mice, male andfemale, were used. ModRNAs are synthesized by in vitro transcription asdescribed above. Modified nucleotides (Trident) are pseudouridine,5-methyl-cytidine, and cap analog. A total of 100-200 μg modified RNAcomplexed with RNAiMax transfection reagent is injected into theperi-infarct region of the myocardium in an open chest surgery postinduction of MI. MRI is performed under light anesthesia (titrated toheart rate and sedation level). LAD ligation and histological analysisis performed as described previously (46.). Three to eight animals usedfor each experiment. For long-term survival, CFW (8-10-week-old) treatedwith CM-specific Luc or Pkm2 modRNAs (n=10) post induction of MI, andwere left to recover for 6 months in the animal facility. Deaths weremonitored and documented over time.

Isolation of Cells from Adult Mice Heart

Hearts are excised and perfused using the Langendorff technique, thecells are processed further by using CD25 specific magnetic beads(dynabeads CD25, Thermo Fisher Scientific) and RNA is isolated fromcells using a RNeasy mini kit (Qiagen). The RNA is further used forRNA-seq and RT-PCR analysis.

Adult Mouse Myocardial Infarction and Heart Failure Models

The MI model described in FIG. 5 was used to test the therapeutic effectafter treatments with CMs-specific Lin28, Pkm2 in CFW or Rosa26^(CD25)mice. The experimental design includes 4 control groups treated with a)vehicle only, b) 100 μg/heart Luc carrying a K-motif modRNA, c) 100μg/heart L7AE modRNA that carry both miR-1 and miR-208 recognitionelements (L7AE miR1+miR208) and d) Luc CMs specific modRNA contain amixture of Luc carrying a K-motif modRNA and L7AE miR1+miR208, 100μg/heart from each modRNA (total 200 μg/heart). Controls groups willserve to assess any unspecific effect of reduction of miR-1 and miR-208in the heart that is not directly related to cell cycle inducerCMs-specific modRNAs. Applicants compared the control groups with 4experimental groups using 100 μg/heart of a) Lin28 and b) Pkm2, carryinga K-motif modRNA and mixture of d) Lin28, and e) Pkm2, carrying aK-motif modRNA with L7AE miR1+miR208 (cell cycle inducer CMs specificmodRNA, 100 μg/heart from each modRNA with (total modRNA 200 μg/heart).We will analyze improved cardiac function after 28 days post MI usingMRI and reduced scar formation, and increased capillary density intohigher rates of long term survival in comparison to control modRNA. Wewill also use the Rosa26Tdtomato mice for lineage tracing model of thetransfected CMs in MI model. Our experimental design includes 1 controlgroup treated with CMs-specific Luc (50 μg/heart) and DD-Cre (50μg/heart) mixed together with 100 μg/heart L7AE miR1+miR208. We willcompare the control groups with 3 experimental groups treated withCMs-specific cell cycle inducer modRNAs. We will use a mixturecontaining DD-Cre (50 μg/heart) and cell cycle inducer gene (Lin28 andPkm2 50 pg/heart) carrying a K-motif modRNA mixed together with 100μg/heart of L7AE miR-1+miR-208 (total modRNA 200 μg/heart). The heart ofRosa26^(Tdtomato) mice, will be directed intramuscular injected withtotal of 100 or 200 μg modRNA. Using CMs lineage tracing model we willtest 28 days post injection transfect CMs size using CMs cross-sectionarea evaluation with anti wheat germ agglutinin (WGA) antibody inimmunofluorescence analysis. We will count the number of transfected CMsper left ventricle and evaluate the number of nuclei per CMs in each ofthe treatments. These testing using Rosa26^(Tdtomato) mice will allow usto evaluate the changes in CMs function after different treatments withCMs specific modRNAs over time.

The MI model described in FIG. 5 was also used for testing geneexpression changes in transfected CMs and non-transfected cells of theleft ventricle. Our experimental design includes 3 control groupstreated with different CMs-specific Luc (50 μg/heart) and inactivate(only extracellular domain) human CD25 (ihCD25, see FIGS. 1 and 8, 50μg/heart) mixed together with 100 μg/heart L7AE miR1+miR208 (totalmodRNA 200 μg/heart). Control groups are compared with 3 experimentalgroups treated with CMs-specific cell cycle inducer modRNAs. Applicantsused a mixture containing ihCD25, (50 μg/heart) and cell cycle inducergene (Lin28 and Pkm2, 50 μg/heart) carrying a K-motif modRNA mixedtogether with 100 μg/heart of L7AE miR-1+miR-208 (total modRNA 200μg/heart). Three days' post treatment with 200 μg CMs-specific ofdifferent Luc controls or cell cycle inducer, Lin28 or Pkm2 in CFW mice(n=10), mice will be sacrificed and hearts will be dissociated withcollagenase. Transfected CMs will be isolated from cardiac cellsuspension using our CMs-specific modRNAsorting approach. This sortingapproach is based on the use of CMs-specific modRNA ihCD25 andcommercially available anti hCD25 magnetic beads (Thermo Fisher).Magnetic beads isolation is been used for variety of application,including cell sorting, for over 30 years. The magnetic beads that beenused for cell sorting are pre-coupled with antibody that can recognizecell surface gene. As transfected CMs usually don't carry a specificcell surface genes, we use the truncated hCD25 to mark the transfectedCMs and allow anti hCD25 magnetic beads to recognize and to attachexclusively to transfected CMs. Using a magnet and elution of residualbeads and un-transfected cells will result in pure isolated transfectedCMs cell population. Immediately after isolation RNA is extracted fromthe sorted transfected CMs and from the eluted fraction of cells(non-transfected cells) and sent for RNA-seq using HIseq2500 system inthe Mount Sinai Genomics Core Facility. Some RNA is used for validationof RNA-seq measurements using Quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR). Downstream targets selection isbased on: a) differentially expressed genes in control Luc vs. modRNAspecifically in CMs; b) overlapping candidates between the differentcell cycle inducer modRNAs treatments; c) Differentially expressed genesin CMs that may influence the gene expression observed innon-transfected cells. For example, up regulation of a relevant receptorin the non-transfected population indicates the secretion of its ligandfrom CMs. d) data mining of the literature. 3-5 targets are selected forvalidation using above approach. To test the hypothesis, we injectedwith CMs-specific nGFP k motif, inactivate (only extracellular domain)human CD25 (ihCD25) mixed together with L7AE miR1-miR208 after MI and wesuccessfully sorted out nGFP and ihCD25 expressing CMs after 24 hrs postinjection FIG. 19.

Magnetic Resonance Imaging (MRI) and Echocardiography (Echo).

CFW mice (8-weeks old) treated with Luc k motif, Luc k motif+miR1-208,miR1-208, Pkm2 k motif and Pkm2 k motif+miR1-208 modRNA were subjectedto MRI assessment on day 28 post LAD ligation.¹¹ We obtaineddelayed-enhancement CINE images on a 7-T Bruker Pharmascan with cardiacand respiratory gating (SA Instruments, Inc, Stony Brook, N.Y.). Micewere anesthetized with 1-2% isoflurane/air mixture. ECG, respiratory,and temperature probes were placed on the mouse, which was kept warmduring scans. Imaging was performed 10 to 20 min after IV injection of0.3 mmol/kg gadolinium-diethylene triamine pentaacetic acid. A stack ofeight to ten short-axis slices of the heart spanning the apex to thebase were acquired with an ECG-triggered and respiratory-gated FLASHsequence with the following parameters: echo time (TE) 2.7 msec withresolution of 200 μm×200 μm; slice thickness of 1 mm; 16 frames per R-Rinterval; 4 excitations with flip angle at 60°. Ejection fraction wascalculated as the difference in end-diastolic and end-systolic volumes,divided by the end-diastolic volume. MRI acquisition and analyses wereperformed blinded to treatment groups. For Echo evaluation of leftventricular systolic function a GE cares in site (V7R5049) equipped witha 40 MHz mouse ultrasound probe were used. Fractional shortening wascalculated based on end diastolic and end systolic dimensions obtainedfrom M-mode ultrasound. Echocardiograms were performed on 6-8hearts/treatment groups.

RNA Isolation and Gene Expression Profiling Using Real-Time PCR

Total RNA was isolated using the RNeasy mini kit (Qiagen) and reversetranscribed using Superscript III reverse transcriptase (Invitrogen),according to the manufacturer's instructions. Real-time qPCR analyseswere performed on a Mastercycler realplex 4 Sequence Detector(Eppendorf) using SYBR Green (Quantitect™ SYBR Green PCR Kit, Qiagen).Data were normalized to 18s expression, where appropriate (endogenouscontrols). Fold-changes in gene expression were determined by the ∂∂CTmethod and were presented relative to an internal control. PCR primersequences are shown in Supplemental Table 3.

TABLE 3 Gene Forward (SEQ ID NO:) Reverse (SEQ ID NO:) Pkm2gtctggagaaacagccaagg (11) cggagttcctcgaatagctg (12) Tnnt2ctgagacagaggaggccaac (13) ttccgctctgtcttctggat (14) Mhy6cagaacaccagcctcatcaa (15) cccagtacctccgaaagtca (16) Pecam1ctgccagtccgaaaatggaac (17) cttcatccaccggggctatc (18) Cdh5attgagacagaccccaaacg (19) ttctggttttctggcagctt (20) αSMAaagctgcggctagaggtca (21) ccctccctttgatggctgag (22) WT1agacacacaggtgtgaaacca (23) atgagtcctggtgtgggtct (24) Mycaggcagctctggagtgagag (25) cctggctcgcagattgtaag (26) Hif1agggtacaagaaaccacccat (27) gaggctgtgtcgactgagaa (28) Pdk1accaggacagccaatacaag (29) cctcggtcactcatcttcac (30) Cdc20ttcgtgttcgagagcgatttg (31) accttggaactagatttgccag (32) Cdk1tttcggccttgccagagcgtt (33) gtggagtagcgagccgagcc (34) Ccnd2gtcacccctcacgacttcat (35) ttccagttgcaatcatcgac (36) Ccnb1aaggtgcctgtgtgtgaacc (37) gtcagccccatcatctgcg (38) 18sagtccctgccctttgtacaca (39) cgatccgagggcctcacta (40) HDac4aaccttagtggggtgctgtg (41) aaggcacaaactcgcatctt (42) Hand2ccagctacatcgcctacctc (43) tggttttcttgtcgttgctg (44) Meox2cacagtgcctgaaatcacca (45) ctggctgtgtttgtcaatgg (46) Gata4tccagcctgaacatctaccc (47) ccatagtcaccaaggctgct (48) Mstntggctcctactggacctctc (49) tgccttttaagatgcagcag (50) MYHCcagaacaccagcctcatcaa (51) gctccttcttcagctcctca (52)

Lineage Tracing in R26^(mTmG) Mice

Rosa26^(mTmG) mice were obtained from the Jackson Laboratory. Allexperiments were performed on age- and sex-matched mice with equal ratioof male and female mice. Healthy mice were chosen randomly from theexpansion colony for each experiment. In this mice line,membrane-targeted tdTomato is expressed under the control of ubiquitouspromoter on Rosa26 locus, whereas membrane-targeted eGFP becomes activeafter Cre-mediated excision of floxed tdTomato. CM-specific Cre modRNA(Cre K-motif+miR1-miR208) was used to exclusively express Cre intransfected CMs. This allowed for lineage tracing of the transfected CMsand their progeny long after the modRNA expression was diminished (>10days). Rosa26mTmG mice were genotyped by PCR with tail DNA as describedin the Jackson Laboratory Genotyping Protocols. Primer sequences are asfollows: Rosa26mT/mG, wild type forward, 5′ CTCTGCTGCCTCCTGGCTTCT-3′(SEQ ID NO: 53), wild type reverse, 5′-CGAGGCGGATCACAAGCAATA-3′ (SEQ IDNO:54), and mutant reverse, 5′-TCAATGGGCGGGGGTCGTT-3′ (SEQ ID NO: 55).In this model, we measured the transfection level of CM-specific CremodRNA, CMs size and number, and the number of nuclei in CMs posttransfection with CM-specific Luc or Pkm2 modRNAs.

Neonatal Rat and Adult Mouse CMs Isolation

CMs from 3-4 day old neonatal rat's heart were isolated as previouslydescribed.¹ Neonatal rats' ventricular CMs were isolated from 4 day-oldSprague Dawley rats (Jackson). We used multiple rounds of digestion with0.14-mg/mL collagenase II (Invitrogen). After each digestion, thesupernatant was collected in Horse serum (Invitrogen). Total cellsuspension was centrifuged at 1500 rpm for 5 min. Supernatants werediscarded and cells were resuspended in DMEM (GIBCO) medium with 0.1 mMascorbic acid (Sigma), 0.5% Insulin-Transferrin-Selenium (100×),penicillin (100 U/mL) and streptomycin (100 μg/mL). Cells were plated inplastic culture dishes for 90 min until most of the non-myocytesattached to the dish and myocytes remained in suspension. Myocytes werethen seeded at 1×10⁵ cells/well in a 24 well plate. Neonatal rat CMswere incubated for 48 hours in DMEM medium containing 5% horse serumplus Ara c. After incubation, cells were transfected with differentdoses of different modRNAs as described in the text. Adult CMs wereisolated from CFW mice after 28 days post MI and modRNA injection usingLangendorff's method as previously described. For CMs count, we averaged3 different counts/sample and 3 hearts/group using a hemocytometer. Thetotal number of CMs counted was approximately 150-200 CMs/aliquot (10 ulaliquots samples using a wide-bore pipette from the total volume of CMsobtained following digestion). The cultured CMs were stained withα-actinin (CMs, Red) antibody (abcam) and Hoechst 33342 for nucleicounts. For nuclei count, approximately 1×10³CMs were counted persample, using 3-4 independent samples per group. nuclei count wasplotted as percentage of counted CMs. For isolation of transfected adultCMs and RNA isolation please see FIGS. 17A-17C.

Mouse MI Model and Histology

All surgical and experimental procedures with mice were performed inaccordance with protocols approved by Institutional Animal Care and UseCommittees at Icahn School of Medicine at Mount Sinai InstitutionalAnimal Care and Use Committee (IACUC) and the MSSM Center forComparative Medicine and Surgery (CCMS). CFW, R26^(mTmG) mice (6-8 weeksold) were anesthetized with isoflurane. MI was induced by permanentligation of the LAD, as previously described³. Briefly, the leftthoracic region was shaved and sterilized. After intubation, the heartwas exposed through a left thoracotomy. A suture was placed to ligatethe LAD. The thoracotomy and skin were sutured closed in layers. Excessair was removed from the thoracic cavity, and the mouse was removed fromventilation when normal breathing was established. In order to determinethe effect of modRNA on cardiovascular outcome after MI, modRNAs(100-150 μg/heart) were injected into the infarct zone immediately afterLAD ligation. The peri-infarct zone near the apex was either snap-frozenfor RNA isolation and subsequent real-time qPCR studies, or fixed in 4%PFA for cryo-sectioning and immunostaining. In all experiments, thesurgeon was blinded to the treatment group. For assessment of hearthistology, hearts were collected at the end of each study. The heartswere excised, briefly washed in PBS, perfused with perfusion buffer,weighted and fixed in 4% PFA at 4° C. overnight. On the next day heartswere washed with PBS and incubated overnight in 30% sucrose. Next,hearts were put in OCT, were frozen and stored at −80° C. The heartblocks were transverse sectioned at 8-9 μm using cryostat. The slideswere further processed for evaluation using immunostaining (see below)or histological scar staining using Masson's trichrome staining kit(Sigma) and were performed according to standard procedures. Measuringratio of heart-weight to body-weight was done using a scale. The ratiowas measured at the end point of each experiment. This ratio wascalculated as the heart tissue weight relative to the mouse totalbody-weight in grams (g).

Immunostaining of Heart Sections Following modRNA Treatment

The mouse hearts were harvested and perfused using perfusion buffer and4% paraformaldehyde (PFA). Hearts were fixed in 4% PFA/PBS overnight onshaker and then washed with PBS for 1 hr and incubated in 30%sucrose/PBS at 4° C. overnight. The next day, hearts were fixed in OCTand frozen at −80° C. Transverse heart sections (8-10 μM) were made bycryostat. Frozen sections were rehydrated in PBS for 5 min followed bypermeabilization with PBS with 0.1% triton X100 (PBST) for 7 min. Slideswere then treated with 3% H₂O₂ for 5 min. After 3 washes with PBST for 5minutes each, the samples were blocked with PBS+5% Donkey normalserum+0.1% Triton X100 (PBSST) for 2 hours at room temperature andprimary antibodies diluted in PBSST were added. Slides were thenincubated overnight at 4° C. Slides were washed with PBST (5 times for 4minutes each) followed by incubation with a secondary antibody(Invitrogen, 1:200) diluted in PBST for 2 hours at room temperature. Thesamples were further washed with PBST (3 times for 5 min each) andstained with Hoechst 33342 (1 μg/m I) diluted in PBST for 7 min. After 5washes with PBST for 4 minutes each, and one time with tap water (for 4minutes), slides were mounted with mounting medium (VECTASHIELD) forimaging. Stained slides were stored at 4° C. All staining were performedon 3-8 hearts/group, with 2-3 sections/heart. In the case ofimmunostaining with wheat germ agglutinin (WGA) for CMs sizequantification, images at 20× magnification were captured and ImageJ wasused to determine the area of each cell. Quantitative analyses involvedcounting of multiple fields from 3-6 independent hearts per group, and 3sections/heart (˜50 cells per field assessed, to a total ˜250 cells persample). For BrdU immunostaining, BrdU (1 mg/ml, Sigma) was added to thedrinking water of adult mice (2-3-month-old) for 7-10 days beforeharvesting the hearts. Quantitative analyses involved counting BrdUpositive CMs in multiple fields from three independent samples pergroup, and 3 sections/heart. The total number of CMs counted was˜1-2×10³ CMs per section. TUNEL immunostaining, was performed accordingto manufacturer's recommendations (In-Situ Cell Death Detection Kit,Fluorescein, Cat #11684795910, Roche). For Immunostaining of neonatalCMs following modRNA treatment, modRNA-transfected neonatal CMs werefixed on coverslips with 3.7% PFA for 15 min at room temperature.Following permeabilization with 0.5% Triton X in PBS for 10 min at roomtemperature, cells were blocked with 5% normal goat/Donkey serum+0.5%Tween 20 for 30 minutes. Coverslips were incubated with primaryantibodies (see supplemental Table 1) in humid chamber for 1 hour atroom temperature followed by incubation with corresponding secondaryantibodies conjugated to Alexa Fluor 488, Alexa Fluor 647 and AlexaFluor 555, and Hoechst 33342 staining for nuclei visualization (all fromInvitrogene). The fluorescent images were taken on Zeiss fluorescentmicroscopy at 10×, 20× and 40× magnification.

Live Cell Imaging of Isolated Rat Neonatal Cardiomyocytes

The time-lapse images of isolated rat neonatal cardiomyocytes posttransfection with nGFP CMs specific modRNA or co-transfected with nGFPand CM-specific Pkm2 modRNAs were acquired with a 10× objective lensevery 10 sec with a confocal spinning disk microscope (Zeiss) following24 hours of time-lapse acquisition.

Statistical Analysis

Statistical significance was determined by paired t-test for the MRIresults, Log-rank (Mantel-Cox) test for survival curves or Student'st-test or One-way ANOVA, Bonferroni post hoc test for other experimentsas detailed in the respective figure legends, with *p<0.05 or lowerconsidered significant. All graphs represent average values, and valueswere reported as mean±standard error of the mean. Two-sided Student'st-test was based on assumed normal distributions. For the quantificationof the number of CD31 luminal structure, WGA, CD45, CD3, TUNEL, BrdU⁺,ki67⁺, pH3⁺ or Aurora B⁺ CMs, the results acquired from at least 3 heartsections.

TABLE 2 Stock concentration Final (mM)/stock amount How much to addconcentration Nucleotide (μmoles) (μl) (mM) ARCA 10 μmoles Use entireTrilink 6 vial GTP 75 mM 36 μl from Ambion 1.5 kit ATP 75 mM 183 μl fromAmbion 7.5 kit CTP 75 mM 183 μl from Ambion 7.5 kit 1.1-methyl 100 mM138 μl from Trilink 7.5 pseudouridine vial Nuclease Free N/A 205 μl from— Water Ambion kit

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1. A method of expressing a protein of interest in a cell, comprising:contacting the cell with a first ribonucleic acid and a secondribonucleic acid, wherein the first nucleic acid comprises a microRNA(miR) recognition element near its 3′UTR that specifically an miRexpressed in the cells and encodes a translation suppressor protein, andthe second nucleic acid comprises a suppressor protein interaction motifthat binds the translation suppressor protein and encodes the protein ofinterest.
 2. The method of claim 1, wherein one or both of the firstribonucleic acid and the second ribonucleic nucleic acid comprisemodified RNA.
 3. The method of claim 1, wherein the cell is acardiomyocyte and the miR comprises one or both of miR1 and miR208. 4.The method of claim 1, wherein the translation suppressor protein isL7Ae and the suppressor protein interaction motif is a k-motif.
 5. Themethod of claim 1, wherein the protein of interest is a reporter proteinor selection marker.
 6. (canceled)
 7. The method of claim 5, wherein thereporter protein or selection marker is selected from one or more ofgreen fluorescence protein (GFP), nuclear GFP (nGFP), inactive humanCD25, (ihCD25) and inactive mouse CD25 (imCD25).
 8. The method of claim1, wherein the protein of interest is a cell cycle inducer protein. 9.The method of claim 8, wherein the cell cycle inducer protein isselected from Lin28 and Pkm2.
 10. The method of claim 1, wherein saidfirst nucleic acid comprises the nucleotide sequence of SEQ ID NO: 2,SEQ ID NO: 3, or SEQ ID NO:
 4. 11. The method of claim 1, wherein saidsecond nucleic acid comprises the nucleotide sequence of SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO:
 8. 12. (canceled) 13.(canceled)
 14. the method of claim 2, wherein the modified RNA comprisesone or more of substitution of one or more uridine with pseudouridine,substitution of one or more cytidine with 5-methylcytidine, and an antireverse cap analog cap substituted at a 5′ end of the ribonucleic acid.15. (canceled)
 16. (canceled)
 17. (canceled)
 18. (canceled) 19.(canceled)
 20. (canceled)
 21. (canceled)
 22. (canceled)
 23. A method ofstimulating proliferation of cardiomyocytes in a subject, comprisingadministering to the subject a first ribonucleic acid and a secondribonucleic acid, wherein the first nucleic acid comprises a microRNA(miR) recognition element near its 3′UTR that specifically binds one orboth of miR-1 and miR-208 and encodes a translation suppressor protein;and the second ribonucleic acid comprises a suppressor proteininteraction motif that binds the translation suppressor protein andencodes a cell cycle inducer protein, wherein the subject suffered amyocardial infarction or suffers from heart failure.
 24. The method ofclaim 23, wherein one or both of the first ribonucleic acid and thesecond ribonucleic acid comprises modified RNA.
 25. The method of claim23, wherein the translation suppressor protein is L7Ae and thesuppressor protein interaction motif is a k-motif.
 26. The method ofclaim 23, wherein the cell cycle inducer protein is selected from Lin28and Pkm2.
 27. The method of claim 23, wherein said first nucleic acidcomprises the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQID NO:
 4. 28. The method of claim 23, wherein said second nucleic acidcomprises the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7 or SEQ ID NO:
 8. 29. The method of claim 23, wherein the modifiedRNA comprises one or more of substitution of one or more uridine withpseudouridine, substitution of one or more cytidine with5-methylcytidine, and an anti reverse cap analog cap substituted at a 5′end of the ribonucleic acid.
 30. A method of treating myocardialinfarction or heart failure in a subject, comprising administering tothe subject a first ribonucleic acid and a second ribonucleic acid,wherein the first ribonucleic acid comprises a microRNA (miR)recognition element near its 3′UTR that specifically binds one or moreof miR-1 and miR-208 and encodes a translation suppressor protein, andthe second ribonucleic acid comprises a k-motif and encodes a cell cycleinducer protein.
 31. The method of claim 30, wherein one or both of thesuppressor protein is L7Ae, and the cell cycle inducer protein isselected from Lin28 and Pkm2.